Most cells in the human body are functionally polarized to generate different regions of the plasma membrane having distinct morphologies and protein compositions. Understanding the regulation of this polarity is critical for understanding the cell biology of diseases. For example, cancer metastasis can be initiated when epithelial cells undergo an inappropriate polarity transition to a mesenchymal state. The microfilament-based cell cortex is largely responsible for defining the morphology of plasma membrane domains, as well as modulating their membrane proteome. Our goal is to identify general mechanisms that cells use to generate specific domains. As an accessible model system, we focus on the microfilament-based cytoskeleton of the apical aspect of polarized epithelial cells and how it contributes to the assembly, dynamics and composition of the abundant microvilli that characterize this domain. Ezrin and the ezrin-binding and scaffolding protein EBP50/NHERF1 regulate apical microvilli, as well as many other microfilament-based surface structures, and this proposal is to understand their individual and collective functions. Ezrin is an abundant, conformationally regulated microfilament-membrane linking protein of microvilli that performs an essential function in defining the apical domain. Our recent work has uncovered two unexpected aspects governing the regulation of microvilli on epithelial cells. First, we have shown that restriction of microvilli to the apical domain requires local ezrin phosphocycling mediated by apically localized LOK/SLK kinases and delocalized phosphatases. Second, we have found that the in vivo dynamics of EBP50 is highly regulated by association of proteins with its PDZ domains. This application focuses on how these dynamic systems are mediated and contribute to the structure and composition of the apical membrane. In aim one we build on our preliminary studies with LOK showing that it has unusual properties for a kinase including its association with phosphorylated substrate, suggesting a positive feedback loop for local activation. We propose to define the mechanism by which LOK is regulated to phosphorylate ezrin, and also to identify the mechanism by which LOK is targeted to, and activated at, the apical membrane. In our second aim we will investigate the mechanism underlying the regulation of EBP50 dynamics. Ezrin and EBP50 are known to regulate the plasma membrane abundance of specific membrane proteins, but to date no study has explored the generality of this regulation. Therefore, in our third aim, we use unbiased proteomics to define the apical membrane proteome in cells lacking microvilli as a result of loss or misregulation of ezrin, and in cells in which the dynamics of scaffolding protein EBP50 is altered. Overall, our studies will provide an unprecedented view of how cells regulate the structure and composition of a specific membrane domain. These studies have relevance to many diseases. For example, EBP50 regulates the abundance of apical CFTR and EGFR, and defects in this regulation of these proteins can cause cystic fibrosis or cancer, respectively.